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The SUVR2 associated proteins FRINGE1 and 2 are required for RdDM in Arabidopsis [Bisulfite-seq]

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ABSTRACT: DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes MET1, CMT3, DRM2 and CMT2. However, new methylation patterns are established exclusively by the enzyme DRM2, which acts within a pathway termed RNA-directed DNA methylation (RdDM). Some RdDM components belong to gene families and have partially redundant functions (i.e. DICER LIKE 2, 3 and 4 and IDN2 PARALOG 1 and 2). Traditional screens, which usually affect single genes, usually fail to detect genes if they are redundant, since the loss of one gene is compensated by a related gene. In an effort to circumvent this caveat, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. We report the discovery of two redundant proteins, SNF2-RING-HELICASE1 and 2 (FRINGE1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRINGE1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRINGE1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined our results identify FRINGE1 and FRINGE2 as novel components of the RdDM machinery. One fringe 1/2 sample analyzed.

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Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery. Small RNA profiles of Columbia 0 wild type, frg1-1 frg2-1 double, frg1 to 5 quintuple, suvr2-1, dcl3-1 and drm1-1 drm2-2 double mutants, 3 biological replicates each.

Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery. mRNA profiles of 14 days old seedlings of Columbia 0 wild type and frg1-1 frg2-1 double mutants, 3 biological replicates each.

Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery. mRNA profiles of 14 days old seedlings of Columbia 0 wild type and frg1-1 frg2-1 double mutants, 3 biological replicates each.

Project description:DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and the maintenance of genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and is targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis and Pol-V, which functions in the downstream DNA methyltransferase targeting phase of the RdDM pathway to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methyl H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy. For wild type plants (ecotype Columbia) and RdDM mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. The Col and nrpe1 BS-seq libraries were previously reported (GSE39247) and so are not part of this submission. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of nrpd1 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPD1. Whole-genome bisulfite sequencing and small RNA sequencing was also performed on shh1 mutant plants transformed with the wild-type SHH1 protein-coding construct as well as multiple constructs containing point mutations. For these complementation libraries a separate shh1 mutant and Col control line were sequenced (“complementation replicates”).

Project description:Eukaryotic DNA methylation is found in silent transposable elements and active genes. Nucleosome remodelers of the DDM1/Lsh family are thought to be specifically required to maintain transposon methylation, but the reason for this is unknown. Here, we find that a chromatin gradient that extends from the most heterochromatic transposons to euchromatic genes determines the requirement of DDM1 for methylation maintenance in all sequence contexts. We also show that small RNA-directed DNA methylation (RdDM) is inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. DDM1 and RdDM independently mediate nearly all transposon methylation, which is catalyzed by the methyltransferases MET1 (CG), CMT3 (CHG), DRM2 (CHH) and CMT2 (CHH), and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that the Arabidopsis genome is defined by a heterochromatic continuum that governs the access of DNA methyltransferases and potentially all DNA binding proteins. Examination of DNA methylation, transcription and nucleosomes in Arabidopsis wild-type and/or ddm1, RdDM and DNA methylase mutants.

Project description:RNA silencing is a mechanism for regulating gene expression at the transcriptional and post-transcriptional levels. Its functions include regulating endogenous gene expression and protecting the cell against viruses and invading transposable elements (TEs). A key component of the mechanism is small RNAs (sRNAs) of 21-24 nucleotides (nt) in length, which direct the silencing machinery in a sequence specific manner to target nucleic acids. sRNAs of 24 nt are involved in methylation of cytosine residues of target loci in three sequence contexts (CG, CHG and CHH), referred to as RNA-directed DNA methylation (RdDM). We previously demonstrated that 24 nt sRNAs are mobile from shoot to root in Arabidopsis thaliana. In this study we demonstrated that methylation of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot. Furthermore, we found that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. These findings were made using base-resolution next generation sequencing approaches and genome wide analyses. Specific classes of short TEs are the predominant targets of mobile sRNA-dependent DNA methylation; classes typically found in gene-rich euchromatic regions. Mobile sRNA-regulated genes were also identified. Mechanistically, we demonstrate that mobile sRNA-dependent non-CG methylation is largely independent of the CMT2/3 RdDM pathway but dependent upon the DRM1/DRM2 RdDM pathway. This is in contrast to non-mobile sRNA-dependent DNA methylation, which predominantly depends upon the CMT2/3 RdDM pathway. These data are complementary to the small RNA sequencing data from Arabidopsis root grafts described in Molnar et al (Science, 2010 May 14;328(5980):872-5).

Project description:Cytosine DNA methylation regulates the expression of eukaryotic genes and transposable elements. Methylation is copied by DNA methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Trans-generational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of trans-generational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells – the cell types that comprise pollen – with mutations in the DRM, CMT2 and CMT3 methyltransferases. Overall design: We isolated sperm and vegetative nuclei from pollen of Col-0, and cmt2, cmt3, drm1 drm2 (d1d2), and h1.1 h1.2 (h1) mutant Arabidopsis thaliana plants, respectively. DNA is extracted from the isolated nuclei and used for bisulfite-sequencing.

Project description:DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the siRNA effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for non-coding Pol V RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts. Whole-genome bisulfite sequencing was done for a wildtype line (ecotype Col) as well as various transgenic lines in a drm2 mutant background (ecotype Col). Each transgenic line expressed a version of the DRM2 protein that was either wildtype or carried induced mutations in order to test the function of various domains in the DRM2 protein. Two sets of whole-genome bisulfite were performed (130615 or 131216) and comparisons were mainly done within sets although comparisons can also be done between sets. The drm2 mutant methylome was also analyzed in this study using a previously published whole-genome bisulfite library (GSE39901).

Project description:DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DRM2 controls RNA-directed DNA methylation in a pathway that also involves the plant specific RNA Polymerase V (Pol V). The Arabidopsis genome also encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase DRM3. Here, we show that DRM3 has moderate effects on global RNA-directed DNA methylation and small RNA abundance throughout the genome, and DRM3 protein physically interacts with Pol V. In drm3 mutants, we observe a lower level of Pol V-dependent transcripts, even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts, and shed further light on the mechanism of RNA-directed DNA methylation. For wildtype plants as well as drm3, drm2, and nrpe1 mutants ChIP-seq was carried out using an endogenous NRPE1 antibody given to us by the Craig Pikaard lab. Two biological replicates of ChIP-seq were also carried out using anti-Flag resin on wildtype and drm3 plants carrying a Flag epitope tagged version of NRPE1. Small RNA sequencing was carried out on Col, drm3, drm2, and nrpe1 plants. Finally, whole-genome bisulfite sequencing analysis was carried out on previously published datasets (as detailed below) which were realigned using a newer genome version and mapping protocol. As such the updated processed files are part of this submission. Please note that the drm2, drm3, and nrpe1 mutant libraries used in this study were previously published (GSE39901), as were the 2 other Col replicates used (GSE36129) as below and thus duplicated sample records were created for the convenient retrieval of the complete raw data from SRA; Bisulfite_seq-Col_1 - GSM881756 Bisulfite_seq-Col_2 - GSM1193638 Bisulfite_seq-drm3 - GSM981017 Bisulfite_seq-drm2 - GSM981015 Bisulfite_seq-nrpe1- GSM981040

Project description:In this work, we studied function of rice DDM1 in coordinating DNA methylation, expression of small (sRNA) and long noncoding (lncRNA) RNAs, and nucleosome positioning by high throughput approaches.We show that the ddm1a/1b mutation resulted in ectopic CHH methylation of transposable elements (TE) and repeats. The ectopicCHH methylationwas dependent on DRM2, a DNA methyltransferase involved in sRNA-dependent DNA-methylation (RdDM). The ddm1a/1b mutation resulted in increases of heterochromatic sRNA but decreases of euchromatic sRNA production,induced expression of a set of developmentally regulated heterochromatic lncRNA , and increased nucleosome occupancy of TE-related or silent genes including heterochromatic lncRNA loci. In particular,DDM1-mediated repression ofCentromericRetrotransposons of Rice1 (CRR1) was essential for centromere function. Overall design: BS-seq of ddm1a/1b/drm2 triple mutant; two replicates for smRNA-seq of wt and ddm1a/1b double mutant.